Precautions

Ensuring the accuracy and reliability of vitamin and nutrient analyses requires meticulous attention to pre-analytical factors, proper specimen handling, and awareness of potential interferences

Special Precautions

Patient Preparation
- Fasting: Some nutrient tests require patients to fast for a specified period (e.g., 8-12 hours) before specimen collection
  - Rationale: Fasting minimizes the effects of recent food intake on nutrient levels
  - Examples: Lipid profiles, glucose, iron studies
- Medications: Certain medications can interfere with nutrient tests
  - Rationale: Medications can affect nutrient absorption, metabolism, or excretion
  - Examples
    - Oral contraceptives: Affect vitamin B6 and folate levels
    - Metformin: Affect vitamin B12 levels
    - Cholestyramine: Affect fat-soluble vitamin absorption
  - Action: Inquire about medications and supplements the patient is taking
- Supplements: Inform patients to avoid taking vitamin and mineral supplements for a specified period before testing
  - Rationale: Supplements can falsely elevate nutrient levels
  - Action: Follow the laboratory’s guidelines for supplement restrictions
- Alcohol Consumption: Alcohol can affect nutrient absorption and metabolism
  - Rationale: Chronic alcohol consumption can lead to deficiencies in thiamin, folate, and other nutrients
  - Action: Instruct patients to avoid alcohol consumption for 24-48 hours before testing
- Timing: For some nutrients, the timing of specimen collection can be important due to diurnal variations or postprandial changes
  - Rationale: Iron levels vary throughout the day; lipid profiles are affected by recent food intake
  - Action: Collect specimens at the specified time

Specimen Collection

Collection Tubes
- Use appropriate collection tubes for each nutrient test
  - Rationale: Different tubes contain different anticoagulants and additives that can affect nutrient stability and assay performance
  - Examples
    - EDTA tubes: Commonly used for vitamin B12, folate, and iron studies
    - Serum separator tubes (SST): Used for fat-soluble vitamins
    - Light-protected tubes: Used for light-sensitive vitamins (e.g., vitamin A, vitamin B6)
- Follow the manufacturer’s instructions for tube selection
Order of Draw
- Follow the correct order of draw when collecting multiple tubes
  - Rationale: Prevents cross-contamination from tube additives
  - Action: Consult the CLSI guidelines for the correct order of draw
Technique
- Use proper venipuncture technique
  - Rationale: Minimizes hemolysis and contamination
  - Action: Avoid prolonged tourniquet time and use a gentle technique
Labeling
- Label specimens immediately and accurately
  - Rationale: Prevents misidentification and errors
  - Action: Use two patient identifiers (e.g., name and date of birth)
Volume
- Collect the correct volume of blood
  - Rationale: Insufficient volume can affect assay performance
  - Action: Follow the manufacturer’s instructions for minimum volume requirements

Specimen Processing

Centrifugation
- Centrifuge specimens promptly after collection
  - Rationale: Separates serum or plasma from cells
  - Action: Follow the manufacturer’s instructions for centrifugation speed and time
Separation
- Separate serum or plasma from cells within the specified time frame
  - Rationale: Prevents hemolysis and degradation of nutrients
  - Action: Follow the manufacturer’s instructions for separation time
Aliquotting
- Aliquot specimens into appropriate containers
  - Rationale: Prevents contamination and allows for multiple tests
  - Action: Use clean, dry containers
Storage
- Store specimens at the appropriate temperature
  - Rationale: Preserves nutrient stability
  - Examples
    - Refrigeration (2-8°C): For short-term storage
    - Freezing (-20°C or -80°C): For long-term storage
- Follow the manufacturer’s instructions for storage conditions
Thawing
- Thaw specimens properly before testing
  - Rationale: Prevents degradation of nutrients
  - Action: Thaw at room temperature or in a refrigerator, and mix gently
Light Protection
- Protect light-sensitive vitamins from light exposure during collection, processing, and storage
  - Rationale: Light can degrade certain vitamins (e.g., vitamin A, vitamin B6, folate)
  - Action: Use amber-colored tubes or wrap clear tubes in aluminum foil

Troubleshooting

High Nutrient Results
- Pre-analytical
  - Patient not fasting
  - Recent supplement intake
  - Medication interference
  - Improper collection tube
  - Delayed processing
  - Contamination of specimen
- Analytical
  - Incorrect calibration
  - Reagent deterioration
  - Interfering substances
  - Cross-reactivity
  - Non-specific binding
Low Nutrient Results
- Pre-analytical
  - Improper collection tube
  - Delayed processing
  - Improper storage
  - Medication interference
- Analytical
  - Incorrect calibration
  - Reagent deterioration
  - Interfering substances
  - Assay drift
Inconsistent Results
- Pre-analytical
  - Patient variability
  - Specimen handling errors
- Analytical
  - Assay variability
  - Interfering substances
  - Cross-reactivity
- Post-analytical
  - Transcription errors
  - Reporting errors
Quality Control Failures
- Pre-analytical
  - QC material improperly stored
  - QC material expired
- Analytical
  - Incorrect calibration
  - Reagent deterioration
  - Instrument malfunction
- Action: Investigate the cause of the failure and take corrective action

Interfering Substances

Hemolysis
- Interference: Releases intracellular components that can interfere with nutrient assays
- Mechanism: Interferes with spectrophotometric or immunoassay measurements
- Mitigation: Avoid hemolysis during collection and processing
Lipemia
- Interference: Turbidity from high lipid concentrations can interfere with spectrophotometric assays
- Mechanism: Lipids scatter light, leading to inaccurate absorbance measurements
- Mitigation: Use lipemia clearing techniques or consider methods less affected by turbidity
Bilirubin
- Interference: High bilirubin levels can interfere with spectrophotometric assays
- Mechanism: Bilirubin absorbs light at similar wavelengths, affecting absorbance readings
- Mitigation: Use methods less susceptible to bilirubin interference or employ bilirubin correction techniques
Drugs
- Interference: Certain drugs can directly affect nutrient levels or interfere with nutrient assays
- Examples
  - Metformin: Affects vitamin B12 levels
  - Cholestyramine: Affects fat-soluble vitamin absorption
  - Oral contraceptives: Affect vitamin B6 and folate levels
- Mitigation: Be aware of patient’s medication list
Heterophile Antibodies
- Interference: Bind to assay antibodies, causing falsely high or low results
- Mechanism: Non-specific binding to assay components
- Mitigation: Use blocking reagents or heterophile antibody blocking tubes
Cross-Reactivity
- Interference: Assay antibodies may cross-react with structurally similar compounds, leading to inaccurate results
- Mechanism: Antibodies bind to unintended targets
- Mitigation: Use highly specific antibodies and be aware of potential cross-reactants
Biotin
- Interference: High levels of biotin can interfere with biotin-streptavidin assays, causing falsely high or low results
- Mechanism: Biotin competes with the analyte for binding to streptavidin
- Mitigation: Ask patients to discontinue biotin supplements for several days before testing
Ascorbic Acid (Vitamin C)
- Interference: Can interfere with certain assays by acting as a reducing agent
- Mitigation: Use methods less susceptible to ascorbic acid interference
Homocysteine
- Interference: Elevated homocysteine levels can interfere with folate and vitamin B12 assays
- Mitigation: Consider measuring homocysteine levels separately

Key Terms

Pre-Analytical: Processes that occur before the sample is analyzed
Analytical: Processes involved in analyzing the sample
Post-Analytical: Processes that occur after the sample is analyzed
Interfering Substance: A substance that affects the accuracy of a test
Hemolysis: The breakdown of red blood cells
Lipemia: The presence of excess lipids in the blood
Calibration: Adjusting an instrument to ensure accurate readings
Quality Control: Samples used to monitor the accuracy and precision of a test
Heterophile Antibodies: Antibodies that bind to multiple antigens
Cross-Reactivity: The ability of an antibody to bind to multiple antigens
Fasting: Abstaining from food or drink for a specified period
Supplement: A product taken orally that contains one or more ingredients intended to supplement the diet

Principles

Result Interpretation